Cortactin在紫绀型先心病内源性肺保护中的作用及机制研究

To maintain a relatively normal lung function under preoperative chronic hypoxia condition, the body of cyanotic congenital heart disease (CHD) patient may undergo a series of compensatory changes. Heart surgery under cardiopulmonary bypass (CPB) is preferred treatment of cyanotic CHD. However, the postoperative lung ischemia reperfusion injury (LIRI) is severe and inevitable. . We previously found that cortactin was up-regulated in the lung of cyanotic mice, while in the subsequent LIRI process its expression decreased with its upstream regulatory protein caveolin-1 expression increasing. Therefore, we hypothesize that decreased cortactin due to up-regulation of caveolin-1 during CPB related LIRI may induce lung injury.. In this project, we will construct cortactin and caveolin-1 gene silencing and overexpression human pulmonary microvascular endothelial cells (HPMVECs) model via siRNA and gene transfer techniques, as well as chronic hypoxic model of mice. First, we will make it explicit the endogenous pulmonary protective role of cortactin in cyanotic CHD. Second, we will clarify the possible underlying mechanism of cortactin down-regulation induced lung injury and the key role of Rac1 during LIRI period. Third, we will explore whether the regulation of caveolin-1-Rac-1-cortactin pathway could promote the continuous overexpression of cortactin and therefore alleviate LIRI after cyanosis in the ischemia reperfusion model of chronic hypoxia mice. Our research will provide a new therapeutic strategy for the prevention and treatment of LIRI for cyanotic CHD.

紫绀型先心病慢性缺氧，机体代偿性改变，术前肺功能无明显受损；但体外循环心脏术后肺缺血再灌注损伤(LIRI)严重。.我们前期组学研究发现，慢性缺氧小鼠肺组织皮层肌动蛋白结合蛋白(cortactin)上调，但随后LIRI期表达降低，并伴caveolin-1水平上升；结合文献，我们推测cortactin是内源性肺保护因子，LIRI期间caveolin-1抑制cortactin表达引起肺损伤。.本课题将以siRNA和基因转移技术①研究cortactin对人肺微血管内皮细胞骨架蛋白和通透性的影响，明确cortactin内源性肺保护作用②证实caveolin-1对cortactin表达的调节作用，Rac1是其中关键环节③研究调控caveolin-1-Rac1-cortactin途径能否减轻LIRI。本课题旨在阐明通过Rac1激活内源性保护因子cortatin是紫绀型先心病术后LIRI新的潜在治疗靶点。

目的：紫绀型先心病患儿术前处于长期的慢性缺氧状态，术后往往更容易出现肺缺血再灌注损伤，严重影响了该类患者的预后。因此希望可以通过本研究明确以下三个问题：1、术前低氧是否是导致紫绀型先心病患者预后较差的危险因素？2、术前长期慢性低氧导致心脏术后肺缺血再灌注损伤加重的具体表现有哪些？3、长期慢性低氧导致CIRBP下调，是否是导致CPB后肺通透性升高并加重肺缺血再灌注损伤的机制？..方法与结果：本研究首先对先心病患者的临床数据进行了回顾性分析，按照是否出现机械通气时间延长及不良预后对患者进行分组，并通过多因素Logistic回归及倾向性评分匹配进行分析，结果表明术前长期慢性低氧是导致术后带管时间延长以及不良预后增加的独立危险因素。接着，将动物和细胞分为常氧组和低氧组，分别构建CPB模型和缺氧复氧模型来模拟体外循环打击，观察两组术后肺损伤的程度，结果表明术前长期慢性低氧会导致肺组织凋亡增加，病理损伤加重，肺水肿加重；肺血管内皮和肺泡上皮通透性增高以及细胞骨架紊乱。最后，为明确低氧导致肺损伤加重的机制，我们对常氧组与低氧组大鼠肺组织进行了蛋白组学分析，结果表明长期慢性低氧会导致CIRBP下降；为进一步明确CIRBP下调是否是导致肺损伤加重的关键蛋白，我们对CIRBP进行了基因敲除并对比了野生型大鼠和基因敲除大鼠CPB后肺损伤的程度，结果表明CIRBP敲除后肺组织细胞凋亡明显增加；炎性细胞聚集、肺间质渗出液增多、肺泡萎缩严重；通透性相关蛋白表达明显降低。..结论：长期慢性低氧是导致紫绀型先心病患儿术后肺损伤加重的重要原因之一，主要表现为肺组织凋亡增加，肺通透性增加，肺水肿加重。这可能是由于低氧导致CIRBP表达降低，从而损伤肺通透性所致。